Method for bisulfite treatment

ABSTRACT

The present application is directed to a method for performing a bisulfite reaction to determine methylation positions in a nucleic acid, i.e. methylated and non-methylated cytosines, whereby the nucleic acid is incubated in a solution comprising the nucleic acid for a time period of 1.5 to 3.5 hours at a temperature between 70 and 90° C., whereby the concentration of bisulfite in the solution is between 3 M and 6.25 M and whereby the pH value of the solution is between 5.0 and 6.0 whereby the nucleic acid, i.e. the cytosine bases in the nucleic acid, are deaminated. Then the solution comprising the deaminated nucleic acid is desulfonated and preferably desalted. The application is further related to a solution comprising bisulfite with a certain pH and uses thereof as well as a kit comprising the solution.

BACKGROUND OF THE INVENTION

This application claims the benefit of priority under 35 U.S.C. §119 ofPCT/EP2004/000729 filed Jan. 28, 2004, and EP Application No. 03001854.3filed Jan. 29, 2003 and EP Application 03010020.0 filed May 2003, thecontents of which are hereby incorporated by reference.

FIELD OF THE INVENTION

The present application is directed to a method for performing abisulfite reaction to determine methylation positions in a nucleic acid,i.e. methylated and non-methylated cytosines, whereby the nucleic acidis incubated in a solution comprising the nucleic acid for a time periodof 1.5 to 3.5 hours at a temperature between 70 and 90° C., whereby theconcentration of bisulfite in the solution is between 3 M and 6.25 M andwhereby the pH value of the solution is between 5.0 and 6.0 whereby thenucleic acid, i.e. the cytosine bases in the nucleic acid, isdeaminated. Then the solution comprising the deaminated nucleic acid isdesulfonated and preferably desalted. The application is further relatedto kit with a solution comprising bisulfite with a certain pH and usesthereof as well as a kit comprising the solution.

BACKGROUND OF THE INVENTION

Genes constitute only a small proportion of the total mammalian genome,and the precise control of their expression in the presence of anoverwhelming background of noncoding deoxyribonucleic acid (DNA)presents a substantial problem for their regulation. Noncoding DNA,containing introns, repetitive elements, and potentially activetransposable elements requires effective mechanisms for its long termsilencing. Mammals appear to have taken advantage of the possibilitiesafforded by cytosine methylation to provide a heritable mechanism foraltering DNA-protein interactions to assist in such silencing. DNAmethylation is essential for the development of mammals and plays apotential role during aging and cancer. The involvement of methylationin the regulation of gene expression and as an epigenetic modificationmarking imprinted genes is well established. In mammals, methylationoccurs only at cytosine residues and more specifically only on cytosineresidues adjacent to a guanosine residue, i.e. at the sequence CG. Thedetection and mapping of DNA methylation sites are essential stepstowards understanding the molecular signals which indicate whether agiven sequence is methylated.

This is currently accomplished by the so-called bisulfite methoddescribed by Frommer, M., et al., Proc. Natl. Acad. Sci. USA 89 (1992)1827-1831, for the detection of 5-methyl-cytosines. The bisulfite methodof mapping 5-methylcytosine uses the effect that sodium hydrogen sulfitereacts with cytosine but not or only poorly with 5-methyl-cytosine.Cytosine reacts with bisulfite to form a sulfonated cytosine reactionintermediate being prone to deamination resulting in a sulfonated uracilwhich can be desulfonated to uracil under alkaline conditions (see FIG.1). It is common knowledge that uracil has the base pairing behavior ofthymine different to the educt cytosine whereas 5-methylcytosine has thebase pairing behavior of cytosine. This makes the discrimination ofmethylated or non-methylated cytosines possible by e.g. bisulfitegenomic sequencing (Grigg, G., and Clark, S., Bioessays 16 (1994)431-436; Grigg, G. W., DNA Seq. 6 (1996) 189-198) or methylationspecific PCR (MSP) disclosed in U.S. Pat. No. 5,786,146. Basic studieson the reaction of uracil and cytosine derivatives with bisulfite havebeen performed by Shapiro et al., JACS 92 (1970)422-424.

There are various documents addressing specific aspects of the bisulfitereaction.

Hayatsu, H., et al., Biochemistry 9 (1970) 2858-2865 reacted uracil,cytosine or their derivatives with 1 M bisulfite, at a pH value around6, at 37° C. for 24 hours. Hayatsu, H., et al., J. Am. Chem. Soc. 92(1970) 724-726 describe the reaction of cytosine with 3 M bisulfite at apH value around 6 at a temperature of 80° C. for 30 min. Slae andShapiro, J. Org. Chem. 43 (1978) 4197-4200 describe the deamination ofcytidine with 1 M bisulfite around neutral pH at various temperatureswhereby the reaction time is not described. There were no investigationsof the deamination of cytosine or methyl-cytosine in nucleic acids inthese documents.

Paulin, R., et al., Nucl. Acids Res. 26 (1998) 5009-5010 investigate theeffects of urea on the efficiency of bisulfite-mediated sequencing of5-methylcytosine in DNA. The DNA is reacted with 3.44 M bisulfite in thepresence of 5.36 M urea and 0.5 mM hydroquinone, at a pH value of 5.0 ata temperature of 55° C. for 15 hours.

Raizis, A. M., et al., Anal. Biochem. 226 (1995) 161-166 disclose abisulfite method for 5-methylcytosine mapping that minimizes templatedegradation. They investigate a method minimizing template degradationusing 5 M bisulfite solutions in the presence of 100 mM hydroquinone ata pH value of 5 at 50° C. A maximum yield of PCR product was observedafter 4 hours. Other conditions as increased pH and lower temperatureswere also investigated.

Grunau, C., et al., Nucleic Acids Res 29 (2001) e65-5, page 1 to 7,perform a systematic investigation of critical experimental parametersof the bisulfite reaction. They investigate bisulfite solutions of 3.87to 4.26 and 5.2 to 5.69 M at a pH value of 5. Temperatures that weretested are 15, 35, 55, 80, 85 and 95° C. for 1, 4 and 18 hours. DNAdegradation is a problem in these investigations.

Wang, R. Y., et al., Nucleic Acids Res. 8 (1980) 4777-4790 disclose theuse of a 3 M bisulfite solution at a pH value of 5.5 at a temperature of37° C. for various time periods in the bisulfite treatment of DNA. Feil,R., et al., Nucleic Acids Res 22 (1994) 695-696 disclose the use of a3.5 M bisulfite solution at a pH value of 5 at a temperature of 0° C.for 24 hours in the bisulfite treatment of DNA. Clark, S. J., et al.,Nucleic Acids Res 22 (1994) 2990-2997, disclose the use of a 3 to 4 Mbisulfite solution at a pH value of 4.8 to 5.8 at a temperature of 37 to72° C. for 8 to 16 hours in the bisulfite treatment of DNA. Tasheva, E.S., and Roufa, D. J., Mol. Cell. Biol. 14 (1994) 5636-5644 disclose theuse of a 1 M bisulfite solution at a pH value of 5 at a temperature of50° C. for 48 hours in the bisulfite treatment of fragments of genomicDNA. Grigg, G. W., DNA Seq 6 (1996) 189-198 discloses the use of a 3.1 Mbisulfite solution at a pH value of 5 at a temperature of 50° C. for 16hours in the bisulfite treatment of DNA. Komiyama, M., and Oshima, S.,Tetrahedron Letters 35 (1994) 8185-8188 disclose the use of a 1 Mbisulfite solution at a pH value of 5 at a temperature of 37° C. for 4hours in the bisulfite treatment of DNA whereby diethylenetriamine ispresent.

Olek, A., et al., Nucleic Acids Res. 24 (1996) 5064-5066 disclose amethod for bisulfite base sequencing whereby bisulfite treatment andsubsequent PCR steps are performed on material embedded in agarosebeads. A 5 M bisulfite solution at a pH value of 5 at a temperature of50° C. is used for 4 hours in the bisulfite treatment of DNA.

A review of DNA methylation analysis can be found in Oakeley, E. J.,Pharmacol. Ther. 84 (1999) 389-400.

Different additional components in the bisulfite mixture are disclosedby WO 01/98528, WO02/31186 or by Paulin, R., et al., Nucleic Acids Res26 (1998) 5009-5010.

Kits for performing bisulfite treatments are commercially available fromIntergen, now distributed by Serologicals Corporation, Norcross, Ga.,USA, e.g. CpGenome™ DNA modification kit(http://www.serologicals.com/products/int_prod/index.html).

All prior art methods for the bisulfite treatment have disadvantages.Therefore, the problem to be solved by the present invention was toprovide a method which overcomes the disadvantages of the prior artmethods.

SUMMARY OF THE INVENTION

The present invention provides a method for the conversion of a cytosinebase, preferably cytosine bases, in a nucleic acid to an uracil base,preferably cytosine bases, whereby preferably a 5-methyl-cytosine base,preferably 5-methyl-cytosine bases, is not significantlyconverted,comprising the steps of

-   a) incubating a solution comprising the nucleic acid for a time    period of 1.5 to 3.5 hours at a temperature between 70 and 90° C.,    whereby the concentration of bisulfite in the solution is between 3    M and 6.25 M and whereby the pH value of the solution is between 5.0    and 6.0 whereby the nucleic acid is deaminated, and-   b) incubating the solution comprising the deaminated nucleic acid    under alkaline conditions whereby the deaminated nucleic acid is    desulfonated.

Further, the invention provides a solution with a pH value between 5.0and 6.0 and comprising bisulfite in a concentration between 3 M and 6.25M, uses thereof and kits comprising this solution.

As known to the expert skilled in the art and according to theinvention, the term “bisulfite” is used interchangeably for“hydrogensulfite”.

According to the invention the term a “bisulfite reaction”, “bisulfitetreatment” or “bisulfite method” shall mean a reaction for theconversion of a cytosine base, preferably cytosine bases, in a nucleicacid to an uracil base, preferably uracil bases, in the presence ofbisulfite ions whereby preferably a 5-methyl-cytosine base, preferably5-methyl-cytosine bases, is not significantly converted. This reactionfor the detection of methylated cytosines is described in detail byFrommer et al., supra and Grigg and Clark, supra. The bisulfite reactioncontains a deamination step and a desulfonation step which can beconducted separately or simultaneously (see FIG. 1; Grigg and Clark,supra). The statement that 5-methyl-cytosine bases are not significantlyconverted shall only take the fact into account that it cannot beexcluded that a small percentage of 5-methyl-cytosine bases is convertedto uracil although it is intended to convert only and exclusively the(non-methylated) cytosine bases (Frommer et al., supra). The expertskilled in the art knows how to perform the bisulfite reaction, e.g. byreferring to Frommer et al., supra or Grigg and Clark, supra whodisclose the principal parameters of the bisulfite reaction. From Grunauet al., supra, it is known to the expert in the field what variations ofthe bisulfite method are possible. In summary, in the deamination step abuffer containing bisulfite ions, optionally chaotropic agents andoptionally further reagents as an alcohol or stabilizers as hydroquinoneare employed and the pH is in the acidic range. The concentration ofbisulfite is between 0.1 and 6 M bisulfite, preferably between 1 M and5.5 M, the concentration of the chaotropic agent is between 1 and 8 M,whereby preferably guanidinium salts are employed, the pH is in theacidic range, preferably between 4.5 and 6.5, the temperature is between0° C. and 90° C., preferably between room temperature (25° C.) and 90°C., and the reaction time is between 30 min and 24 hours or 48 hours oreven longer, but preferably between 1 hour and 24 hours. Thedesulfonation step is performed by adding an alkaline solution or bufferas e.g. a solution only containing a hydroxide, e.g sodium hydroxide, ora solution containing ethanol, sodium chloride and sodium hydroxide(e.g. 38% EtOH, 100 mM NaCl, 200 mM NaOH) and incubating at roomtemperature or elevated temperatures for several min, preferably between5 min and 60 min.

The method according to the invention allows a relatively short reactiontime of the bisulfite reaction giving the possibility to perform a DNAassay within one working day. One parameter to speed the reaction is thetemperature. To decrease the DNA degradation process, a low pH value isof advantage. By the use of a 5 M bisulfite solution of a pH value of5.5 at a temperature of approximately 80° C., a reaction time of e.g.between 120 and 180 min is possible. Further, the reaction underconditions according to the invention is more specific for cytosinecompared to 5-methylcytosine as with standard conditions after 16 h.Additives for stabilization of the bisulfite reagent like hydroquinoneare possible.

DETAILED DESCRIPTION OF THE INVENTION

The invention is related to a method for the conversion of a cytosinebase, preferably cytosine bases in a nucleic acid to an uracil base,preferably uracil bases, whereby preferably a 5-methyl-cytosine base,preferably 5-methyl-cytosine bases, is not significantly converted,comprising the steps of

-   -   a) incubating a solution comprising the nucleic acid for a time        period of 1.5 to 3.5 hours at a temperature between 70 and 90°        C., whereby the concentration of bisulfite in the solution is        between 3 M and 6.25 M and whereby the pH value of the solution        is between 5.0 and 6.0 whereby the nucleic acid is deaminated,        and    -   b) incubating the solution comprising the deaminated nucleic        acid under alkaline conditions whereby the deaminated nucleic        acid is desulfonated.

In a preferred embodiment of the invention, the method may furthercomprise the step of desalting the solution comprising the deaminatedand desulfonated nucleic acid. This can be achieved e.g. byultrafiltration, gel filtration, precipiation as known to the expertskilled in the art or by binding to magnetic glass particles asdescribed in WO 96/41811.

In a preferred embodiment of the invention, the temperature in themethod according to the invention is between 75 and 85° C. In anotherpreferred embodiment of the invention, the concentration of bisulfite isbetween 3.2 M and 6 M, preferably between 4.75 M and 5.5 M. In anotherpreferred embodiment of the invention, the pH value of the solution isbetween 5.25 and 5.75. In another preferred embodiment of the invention,the time period is between 1.75 and 3 hours. In another preferredembodiment of the invention, the time period is between 2 and 3 hours,preferably between 2 and 2.5 hours. The reaction is also possible in atime period between 0.75 and 3.5 hours. In the most preferred embodimentof the invention, in step a) the temperature is 80° C., theconcentration of bisulfite is 5 M, the pH value of the solution is 5.5and the time period is preferably between 2 and 2.5 or 3 hours, mostpreferred 2 hours.

The method is preferably performed in solution, however, it is alsofeasible that the method according to the invention is performed whilethe nucleic acid is in a solid phase bound form, i.e. it is bound to asolid phase under suitable conditions. The solid phase may be a siliconoxide, preferably in the form of glass fleeces or fibers or magneticglass particles as described in WO96/41811, WO 00/32762 and WO 01/37291.The principal method of performing a bisulfite treatment while thenucleic acid is bound to a solid phase is e.g. described e.g. in theEuropean patent application with the number EP 02 019 097.1 and EP 02028 114.3.

The expert skilled in the art knows how to perform the bisulfitereaction, e.g. by referring to Frommer et al., supra, Grigg and Clark,supra or Grunau et al., supra who disclose the principal parameters ofthe bisulfite reaction.

In an embodiment of the invention, the nucleic acid is deoxyribonucleicacid (DNA), in particular genomic DNA or nucleic acid, i.e. the DNA ornucleic acid which is found in the organism's genome and is passed on tooffspring as information necessary for survival. The phrase is used todistinguish between other types of DNA, such as found within plasmids.The source of the nucleic acid may be eukaryotic or prokarytic,preferably from vertebrates, particularly from mammalians, mostpreferred from animals or humans.

In an embodiment of the invention the nucleic acid is obtained from abiological sample using the solid phases as described above and methodsknown to the expert in the field. The biological sample comprises cellsfrom multicellular organisms as e.g. human and animal cells such asleucocytes, and immunologically active low and high molecular chemicalcompounds such as haptens, antigens, antibodies and nucleic acids, bloodplasma, cerebral fluid, sputum, stool, biopsy specimens, bone marrow,oral rinses, blood serum, tissues, urine or mixtures thereof. In apreferred embodiment of the invention the biological sample is a fluidfrom the human or animal body. The biological sample can be blood, bloodplasma, blood serum, tissue or urine. The biological sample comprisingthe nucleic acids is lysed to create a mixture of biological compoundscomprising nucleic acids and other components. Procedures for lysingbiological samples are known by the expert and can be chemical,enzymatic or physical in nature. A combination of these procedures isapplicable as well. For instance, lysis can be performed usingultrasound, high pressure, shear forces, alkali, detergents orchaotropic saline solutions, or proteases or lipases. For the lysisprocedure to obtain nucleic acids, special reference is made toSambrook, J., et al., in “Molecular Cloning: A Laboratory Manual”(1989), eds. J. Sambrook, E. F. Fritsch and T. Maniatis, Cold SpringHarbour Laboratory Press, Cold Spring Harbour, N.Y.; and Ausubel, F., etal., in “Current protocols in molecular biology” (1994), eds. F.Ausubel, R. Brent and K. R. E., Wiley & Sons, New York. Then the nucleicacids are isolated from the lysis mixture using the methods known to theexpert skilled in the art, e.g. using solid phases as magnetic glassparticles (WO96/41811), and can then be subjected to the methodsaccording to the invention, i.e. the bisulfite treatment according tothe invention. Chaotropic agents are also used to lyse cells to preparea mixture between nucleic acids and other biological substances (seee.g. Sambrook et al. (1989) or EP 0 389 063). Afterwards a materialcomprising glass or silica may be added and a purification effectresults from the behavior of DNA or RNA to bind to material with a glasssurface under these conditions i.e. in the presence of certainconcentrations of a chaotropic agent, higher concentrations of organicsolvents or under acidic conditions. Sequence specific capturing canalso be used for this purpose.

In a preferred embodiment of the invention, the nucleic acid isamplified after the steps of the method according to the invention withthe polymerase chain reaction (PCR: EP 0 201 184; EP-A-0 200 362; U.S.Pat. No. 4,683,202). The amplification method may also be the LigaseChain Reaction (LCR: Wu, D. Y., and Wallace, R. B., Genomics 4 (1989)560-569; and Barany, P., Proc. Natl. Acad. Sci. USA 88 (1991) 189-193),Polymerase Ligase Chain Reaction (Barany, F., PCR Methods Appl. 1 (1991)5-16), Gap-LCR (PCT Patent Publication No. WO 90/01069), Repair ChainReaction (European Patent Publication No. EP-A 0 439 182), 3SR (Kwoh, D.Y., et al., Proc. Natl. Acad. Sci. USA 86 (1989) 1173-1177; Guatelli, J.C., et al., Proc. Natl. Acad. Sci. USA 87 (1990) 1874-1878; PCT PatentPublication No. WO 92/0880A), and NASBA (U.S. Pat. No. 5,130,238).Further, there are strand displacement amplification (SDA), transciptionmediated amplification (TMA), and Q□-amplification (for a review seee.g. Whelen, A. C., and Persing, D. H., Annu. Rev. Microbiol. 50 (1996)349-373; Abramson, R. D., and Myers, T. W., Curr. Opin. Biotechnol. 4(1993) 41-47). Particularly preferred amplification methods according tothe invention are the methylation specific PCR method (MSP) disclosed inU.S. Pat. No. 5,786,146 which combines bisulfite treatment andallele-specific PCR (see e.g. U.S. Pat. No. 5,137,806, U.S. Pat. No.5,595,890, U.S. Pat. No. 5,639,611).

In a preferred embodiment, the method may further comprise the step ofdetecting the amplified nucleic acid. The amplified nucleic acid may bedetermined or detected by standard analytical methods known to theperson skilled in the art and described e.g. by Sambrook, J., et al., in“Molecular Cloning: A Laboratory Manual” (1989), eds. J. Sambrook, E. F.Fritsch and T. Maniatis, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.; Lottspeich and Zorbas, in “Bioanalytik” (1998),eds. L. a. Zorbas, Spektrum Akademischer Verlag, Heidelberg, Berlin,Germany; or by Ausubel, F., et al., in “Current protocols in molecularbiology” (1994), eds. F. Ausubel, R. Brent and K. R. E., Wiley & SonsVerlag, New York. There may be also further purification steps beforethe target nucleic acid is detected e.g. a precipitation step. Thedetection methods may include but are not limited to the binding orintercalating of specific dyes as ethidium bromide which intercalatesinto the double-stranded DNA and changes its fluorescence thereafter.The purified nucleic acids may also be separated by electrophoreticmethods optionally after a restriction digest and visualized thereafter.There are also probe-based assays which exploit the oligonucleotidehybridisation to specific sequences and subsequent detection of thehybrid. It is also possible to sequence the target nucleic acid afterfurther steps known to the expert in the field. Other methods apply adiversity of nucleic acid sequences to a silicon chip to which specificprobes are bound and yield a signal when a complementary sequence binds.

In a particularly preferred embodiment of the invention, the nucleicacid is detected by measuring the intensity of fluorescence light duringamplification. This method entails the monitoring of real timefluorescence. A particularly preferred method exploiting simultaneousamplification and detection by measuring the intensity of fluorescentlight is the TaqMan® method disclosed in WO 92/02638 and thecorresponding U.S. Pat. No. 5,210,015, U.S. Pat. No. 5,804,375, U.S.Pat. No. 5,487,972. This method exploits the exonuclease activity of apolymerase to generate a signal. In detail, the nucleic acid is detectedby a process comprising contacting the sample with an oligonucleotidecontaining a sequence complementary to a region of the target nucleicacid and a labeled oligonucleotide containing a sequence complementaryto a second region of the same target nucleic acid strand, but notincluding the nucleic acid sequence defined by the firstoligonucleotide, to create a mixture of duplexes during hybridizationconditions, wherein the duplexes comprise the target nucleic acidannealed to the first oligonucleotide and to the labeled oligonucleotidesuch that the 3′-end of the first oligonucleotide is adjacent to the5′-end of the labeled oligonucleotide. Then this mixture is treated witha template-dependent nucleic acid polymerase having a 5′ to 3′ nucleaseactivity under conditions sufficient to permit the 5′ to 3′ nucleaseactivity of the polymerase to cleave the annealed, labeledoligonucleotide and release labeled fragments. The signal generated bythe hydrolysis of the labeled oligonucleotide is detected and/ormeasured. TaqMan® technology eliminates the need for a solid phase boundreaction complex to be formed and made detectable. In more generalterms, the amplification and/or detection reaction of the methodaccording to the invention is a homogeneous solution-phase assay.Further preferred methods are the formats used in the LightCycler®instrument (see e.g. U.S. Pat. No. 6,174,670). Particularly preferred isthe use of bisulfite treatment, amplification with or withoutmethylation specific primers in the presence of a methylation-specificprobe and real-time fluorescence detection as described in U.S. Pat. No.6,331,393.

In a preferred embodiment of the present invention, the method isautomated, i.e. the method carries out an automatable process as e.g.described in WO 99/16781. Automatable process means that the steps ofthe process are suitable to be carried out with an apparatus or machinecapable of operating with little or no external control or influence bya human being. Automated method means that the steps of the automatablemethod are carried out with an apparatus or machine capable of operatingwith little or no external control or influence by a human being. Onlythe preparation steps for the method may have to be done by hand, e.g.the storage containers have to filled up and put into place, the choiceof the samples has to be done by a human being and further steps knownto the expert in the field, e.g. the operation of the controllingcomputer. The apparatus or machine may e.g. add automatically liquids,mix the samples or carry out incubation steps at specific temperatures.Typically, such a machine or apparatus is a robot controlled by acomputer which carries out a program in which the single steps andcommands are specified. In a preferred embodiment of the invention, themethod is in a high-throughput format, i.e. the automated methods iscarried out in a high-throughput format which means that the methods andthe used machine or apparatus are optimized for a high-throughput ofsamples in a short time.

Preferably, the method according to the invention is used indiagnostics, for diagnostic analysis or for bioanalytics, or for thescreening of tissue or fluids from the human or even animal body for thepresence of a certain methylation pattern. Further, the method accordingto the invention is used to enhance the speed, accuracy or sensitivityof the detection of methylation sites in nucleic acids.

In another embodiment of the invention, a solution with a pH valuebetween 5.0 and 6.0 and comprising bisulfite in a concentration between3 M and 6.25 M is used in a reaction at a reaction temperature between70 and 90° C. wherein a cytosine base, preferably cytosine bases, in anucleic acid are converted to an uracil base, preferably uracil bases,in the presence of bisulfite ions whereby preferably a 5-methyl-cytosinebase, preferably 5-methyl-cytosine bases, is not significantlyconverted. Preferably, the pH value of the solution is between 5.25 and5.75 and the concentration of bisulfite is between 3.2 M and 6 M,preferably between 4.75 M and 5.5 M. In the most preferred embodiment,the pH value of the solution is 5.5 and the concentration of bisulfiteis 5 M. The solution may also contain hydroquinone for stabilisation.The solution according to the invention is preferably an aqueoussolution. Preferably, the reaction temperature is between 75 and 85° C.

In another embodiment of the invention a kit comprising a solutionaccording to the invention. Preferably, The solution has a pH valuebetween 5.25 and 5.75, more preferably between 5.4 and 5.6, andcomprises bisulfite in a concentration between 3 M and 6.25 M.Preferably, the concentration of bisulfite is between 3.5 M and 6 M,preferably between 4.75 M and 5.5 M. The solution may optionally containhydroquinone. In the most preferred embodiment, the pH value of thesolution is 5.5 and the concentration of bisulfite is 5 M. Such kitsknown in the art further comprise plastics ware which may be used duringthe bisulfite procedure as e.g. microtiter-plates in the 96 or 384 wellformat or reaction tubes manufactured e.g. by Eppendorf, Hamburg,Germany. The kit may further comprise a washing solution which issuitable for the washing step of the solid phase, in particular, theglass fleece or membrane or the magnetic glass particles. Often thewashing solution is provided as a stock solution which has to be dilutedbefore the use. The kit may further comprise an eluent, i.e. a solutionor a buffer (e.g. TE, 10 mM Tris, 1 mM EDTA, pH 8.0) or pure water toelute the DNA or RNA bound to the solid phase. Further, additionalreagents may be present which contain buffers suitable for use in thepresent invention. Preferably, the kit according to the invention isused for a reaction wherein a cytosine base, preferably cytosine bases,in a nucleic acid are converted to an uracil base, preferably uracilbases, in the presence of bisulfite ions whereby preferably a5-methyl-cytosine base, preferably 5-methyl-cytosine bases, are notsignificantly converted.

In another embodiment of the invention, a solution is provided with a pHvalue between 5.4 and 5.6 and comprising bisulfite in a concentrationbetween 3.5 M and 6.25 M. The solution optionally contains hydroquinoneor other radical scavengers. Preferably, the concentration of bisulfiteis between 3.75 M and 6 M, preferably between 4.75 M and 5.5 M. In themost preferred embodiment, the pH value of the solution is 5.5 and theconcentration of bisulfite is 5 M.

The following examples, references, sequence listing and figures areprovided to aid the understanding of the present invention, the truescope of which is set forth in the appended claims. It is understoodthat modifications can be made in the procedures set forth withoutdeparting from the spirit of the invention.

DESCRIPTION OF THE FIGURES

FIG. 1: The steps of the bisulfite method

FIG. 2 to 14: HPLC profiles of the reaction mixtures after certain timeperiods as indicated in the examples

1 EXAMPLES 1.1 Comparison Optimized Conditions with Standard ConditionsUsing a Model System (Oligonucleotide) and Analysis by HPLC

1.1.1 Method:

1.1.1.1 Composition of Reagents

Bisulfite reagent pH = 5,0/50° C.: 1,9 g Na₂S₂O₅ (“Standard”) 2 mlMillipore water 0,7 ml 2M NaOH 0,5 ml 1M hydroquinone (optional)addition of Millipore water to a volume of 4 ml Bisulfite reagent pH =5,5/80° C.: 1,9 g Na₂S₂O₅ 1 ml Millipore water 2 ml 2M NaOH 0,5 ml 1Mhydroquinone (optional) addition of Millipore water to a volume of 4 ml

Hydroquinone can be added optionally; it is not necessary if the reagentis prepared freshly for the experiment.

Sequences:

The oligonucleotides are synthesized using standard automatedsolid-phase synthesis procedure applying phosphoramidite chemistry.

GSTP1 Sequence:

SEQ ID NO:1: 5′-d(GAGGGGCGCCCTGGAGTCCC)-3′ (sense strand) SEQ ID NO:2:5′-d(GGGACTCCAGGGCGCCCCTC)-3′ (antisense strand) SEQ ID NO:3:5′-d(GAGGGGUGUUUTGGAGTUUU)-3′ (sense strand C converted to U (product))SEQ ID NO:4: 5′-d(GGGAUTUUAGGGUGUUUUTU)-3′ (antisense strand C convertedto U (product))C or C^(Me) in the Center Position of T₁₀:

SEQ ID NO:5: 5′-d(T₅CT₅)-3′ SEQ ID NO:6: 5′-d(T₅C^(Me)T₅)-3′ SEQ IDNO:7: 5′-d(T₅UT₅)-3′ SEQ ID NO:8: 5′-d(T₁₁)-3′

1.1.1.2 Reaction Conditions:

Ca. 5 nmole of a single stranded oligonucleotide or 5 nmole of eachstrand of a double stranded oligonucleotide are dissolved in 20 μl ofMillipore water, then 200 μl of the bisulfite reagent are added.Thereafter the reaction tube is placed into a thermomixer (50° C. or 80°C.; 600 rpm). After t=x hours the reaction is stopped by addition of 500μl of 2.5M NaOH (desulfonation). After 30 min at r.t. the reactionmixture is desalted over a Sephadex G25 column. The oligonucleotidecontaining fraction is evaporated and dissolved in 200 μl of Milliporewater to be analyzed by HPLC.

Evaluation Analytical HPLC: column: Dionex DNA Pac PA-100 SEL buffer A:0,01 M NaOH, 0,2 M NaCl buffer B: 0,01 M NaOH, 1M NaCl gradient: 50-100%B in 25 min

Data evaluation: HPLC chromatograms are compared by HPLC-area % of theproduct peak at t=x and are shown in FIGS. 2 to 12

1.1.2 Results

1.1.2.1 Reaction Kinetics GSTP1 ds at T=80° C., pH=5.5

A double determination was performed according to the standard protocoldescribed above with the GSTP1 sequences SEQ ID NO:1 which is the sensestrand and SEQ ID NO:2 which is the antisense strand, the mean averagevalues are calculated.

HPLC area % t[min] product FIG. 10 0 2 30 27,0 3 60 77,5 4 90 87,5 5 12089,9 6 150 90,4 7 180 88,6 8

1.1.2.2 Reaction Kinetics GSTP1 ds at T=50° C., pH =5.0 (“StandardConditions”)

A double determination was performed according to the standard protocoldescribed above with the GSTP1 sequences SEQ ID NO:1 which is the sensestrand and SEQ ID NO:2 which is the antisense strand, the mean averagevalues are calculated.

HPLC area % t[h] product FIG. 1 15,6 9 2 41,2 10 4 72,5 8 89,4 11 1691,8 12 20 85,0

1.1.2.3. Specificity of Bisulfite Reaction T5CMeT5 at T=80° C., pH=5.5(Optimized Conditions) Compared to “Standard Conditions” at T=50° C.,pH=5.0

In order to evaluate the specificity of bisulfite reaction, theoligonucleotide 5′-T₅C^(Me)T₅-3′ (SEQ ID NO: 6) was evaluated under theindicated conditions. The results were as follows:

5 M bisulfite pH 5.0T=50° C./t=16 h (standard conditions) (see FIG. 13for an exemplary chromatogram):

HPLC HPLC ratio HPLC area % area % area % sample T₅C^(Me)T₅ % T₁₁T₁₁/T₅C^(Me)T₅ 1 82,4 5,20 0,0630 2 83,3 5,11 0,00614 3 80,2 5,52 0,06884 80,5 5,92 0,0735 Mean value 81,6 5,44 0,0667

5 M bisulfite pH 5.5/T=80° C./t=2 h (optimized conditions) (see FIG. 14for an exemplary chromatogram)

HPLC HPLC ratio HPLC area % area % area % sample T₅C^(Me)T₅ % T₁₁T₁₁/T₅C^(Me)T₅ 1 89,9 2,65 0,0295 2 89,5 2,46 0,0275 3 90,2 2,24 0,02484 89,7 2,88 0,0322 Mean value 89,8 2,56 0,02851.1.3 Conclusions

Conditions according to the invention lead to a similar product yieldafter 2 h reaction time as standard conditions after 8-16 h. Specificityof bisulfite reaction is significantly better for conditions accordingto the invention after 2 h compared to “standard conditions” after 16 h.

1.2 Comparison of Certain Conditions of the Bisulfite Method Using PCRof Bisulfite Treated Genomic DNA

1.2.1 General

The fact that the bisulfite reaction has worked and convertednon-methylated cytosines to uracil can also be demonstrated by apolymerase chain reaction whereby primers are used which are specific toa region of the nucleic acid sequence wherein non-methylated cytosineshave been converted to uracils, i.e. the base adenine in the primer isopposite to the uracil being the bisulfite reaction product fromnon-methylated cytosines. In case of incomplete conversion, the primercould not hybridize to this region as there would be cytosines notmatching the adenine bases in the primer. This would have the effectthat no PCR product would be obtained.

An improved method to perform rapid polymerase chain reactions isdisclosed e.g. in U.S. Pat. No. 6,174,670 and is used in theLightCycler® instrument (Roche, Mannheim, Germany). In this method, twolabeled probes can come into close proximity in an amplificate dependentmanner so that the two labels can perform a fluorescence energy transfer(FRET). The amount of the amplificate thereby correlates with theintensity of the emitted light of a certain wavelength. This specificPCR method can therefore be used to analyze whether a completeconversion of non-methylated cytosines was obtained, by e.g. analyzingthe promoter region of the glutathion-S-transferase π gene (see e.g.U.S. Pat. No. 5,552,277, Genbank accession code M24485 and Morrow et al.(1989) Gene 75, 3-11) using suitable probes and primers. However, theexpert skilled in the art knows that other methods can be used for thisevaluation as well. Fluorescence measurements are normalized by dividingby an initial fluorescence measurement, i.e., the backgroundfluorescence, obtained during a cycle early in the reaction while thefluorescence measurements between cycles appear to be relativelyconstant. The cycle number chosen for the initial fluorescencemeasurement is the same for all reactions compared, so that allmeasurements represent increases relative to the same reaction cycle. Inthe early cycles of a polymerase chain reaction amplification, thenumber of target molecules can be described by the geometric equationN_(i)=N_(o)×(1+E)¹, where N_(o)=the number of target molecules at thestart of the reaction, N_(i)=the number of target molecules at thecompletion of the i-th cycle, E=the efficiency of the amplification(0≦E≦1). During this geometric growth phase of the amplification, thenumber of cycles required to reach a particular threshold value (C_(T)value or crossing point) is inversely proportional to the logarithm of(1+E). Thus, the C_(T) value represents a measure of the reactionefficiency that allows comparisons between reactions. A decrease in theC_(T) value, which means that the reaction reached the threshold valuein fewer cycles, indicates an increase in reaction efficiency. As theincrease in amplification product is monitored by measuring the increasein reaction fluorescence, the C_(T) is defined herein as the number ofamplification cycles carried out until the fluorescence exceeded anarbitrary fluorescence level (AFL). The AFL was chosen close to thebaseline fluorescence level, but above the range of random fluctuationsin the measured fluorescence, so that the reaction kinetics weremeasured during the geometric growth phase of the amplification.Accumulation of amplified product in later cycles inhibits the reactionand eventually leads to a reaction plateau. An AFL of 1.5 was chosen forall reactions. Because a PCR amplification consists of discrete cyclesand the fluorescence measurements are carried out once per cycle, themeasured fluorescence typically increases from below the AFL to abovethe AFL in a single cycle. To improve the precision of the measurements,an “exact” number of cycles to reach the AFL threshold, referred toherein as the C_(T) value or crossing point, was calculated byinterpolating fluorescence measurements between cycles.

1.2.2 Methods

1.2.2.1 Denaturation of DNA

100 μl of methylated DNA (Intergen, distributed by SerologicalsCorporation, Norcross, Ga., USA; Cat S 7821) dilution (100 ng/assayspiked in 1000 ng human DNA background, Roche Cat.1691112; 4 replicatesper method), and 12 μl 2 M NaOH are mixed and incubated for 15 min at37° C.

1.2.2.2 Deamination of DNA

112 μl of the denatured DNA are mixed with 200 μl bisulfite reagent(2.5M sodium disulfite, 125 mM hydroquinone, pH 5.1) and incubated for20 h at 50° C. (“Standard method”)

or

112 μl of the denatured DNA are mixed with 200 μl bisulfite reagent(2.5M sodium disulfite, 125 mM hydroquinone, pH 5.5) and incubated for 2h at 80° C. (“BIS-METHOD”).

1.2.2.3 Processing Using Magnetic Glass Particles (MGPs)

312 μl of the deaminated DNA (from both methods respectively) are mixedwith 600 μl binding buffer (MagNAPure DNA Isolation Kit I, Roche Cat.Nr. 3 003 990) and 75 μl magnetic glass particle solution (MagNAPure DNAIsolation Kit I) and incubated for 15 min/room temperature withcontinuous mixing in order to bind the nucleic acid to the MGPSaccording to the method described in the European patent applicationswith the numbers EP02019097.1 or EP02028114.3. Thereafter, the magneticglass particles (MGPs) are washed three times with 1 ml 70% ethanol.Bound free separation is done in a magnetic separator (RocheCat.1641794). Thereafter, desulfonation takes place by adding 250 μl 38%EtOH/100 mM NaCl/200 mM NaOH to the DNA bound to the MGPs; the mixtureis incubated for 5 min at room temperature with mixing. Thereafter theMGPs are washed two times with 90% Ethanol. To get rid of ethanol reststhe MGPs were heated for 15 min./60° C. in a thermomixer with open lid.Thereafter the DNA is eluted with 50 μl 10 mM Tris/0.1 mM EDTA pH 7.5(15 min./60° C.). 10 μl of the eluted DNA is used for subsequent PCRanalysis.

1.2.2.4 Detection of the Bisulfite Treated DNA by Using a Specific PCRon the LightCycler® Instrument (Hyprobe-Format)

1.2.2.4.1 Composition of Mastermix

LightCycler® FastStart DNA Master HybridizationProbe 1× (Roche 2239272),2 mM MgCl₂, forward Primer 0.5 μM, reversed Primer 0.5 μM, donor probe250 nM, acceptor probe 250 nM, template 10 μl, total PCR volume 20 μl.

1.2.2.4.2 PCR-Conditions

-   Denaturation 10 min/95° C.-   55 cycles    -   95° C./10 s    -   65° C./10 s—signal acquisition    -   72° C./10 s Ramp time 20° C./s

Samples were run in parallel in the same run on the LightCycler®instrument.

1.2.2.5 Results:

Median Sample-Nr.* BIS-Method Ct-value Ct-value 1 “standard” 30.08 2“standard” 30.07 3 “standard” 30.13 4 “standard” 30.13 30.10 5 “BISMethod” 29.11 6 “BIS Method” 30.14 7 “BIS Method” 30.14 8 “BIS Method”29.58 29.74

The crossing points show that the “BIS Method” according to theinvention is slightly more sensitive as the “standard” method.

1.2.3 Example: Variation of Temperature and Time of the Bisulfite MethodAccording to the Invention

The following experiments were performed using the experimental setup ofthe example under 1.2.1 whereby the temperature and the incubation timewere varied and the indicated ct-values measured.

Incubation time Median Sample [min] Ct-value Temperature Ct-value 1 18028.56 80° C. 28.35 2 180 28.15 80° C. 3 180 28.03 80° C. 4 180 28.64 80°C. 5 150 28.86 80° C. 28.57 6 150 28.64 80° C. 7 150 28.08 80° C. 8 15028.71 80° C. 9 120 28.94 80° C. 28.94 10 120 29.02 80° C. 11 120 28.7780° C. 12 120 29.04 80° C. 13 90 29.76 80° C. 29.67 14 90 29.76 80° C.15 90 29.60 80° C. 16 90 29.57 80° C. 17 60 30.02 95° C. 30.86 18 6029.86 95° C. 19 60 33.54 95° C. 20 60 30.01 95° C.

This experiment shows that the extension of incubation time to 3 hoursis not critical, whereas shortening of the incubation time to 90 min.results in a small loss of sensitivity. A higher loss of sensitivityresulted when the incubation time was shortened to 60 min but incubationtemperature was elevated to 95° C.

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1. Method for the conversion of a cytosine base in a nucleic acid to anuracil base comprising a) incubating a solution comprising the nucleicacid for a time period that is not less than 1.5 hours and further isnot more than 3.5 hours at a temperature between 70 and 90° C., whereinthe concentration of bisulfite in the solution is between 3 M and 6.25 Mand wherein the pH value of the solution is between 5.25 and 5.75,whereby the nucleic acid is deaminated, and b) incubating the solutioncomprising the deaminated nucleic acid under alkaline conditions wherebythe deaminated nucleic acid is desulfonated.
 2. Method according toclaim 1, wherein in step a) the temperature is between 75 and 85° C. 3.Method according to claim 1, wherein the concentration of bisulfite isbetween 3.2 M and 6 M.
 4. Method according to claim 1, wherein the timeperiod is between 1.75 and 3 hours.
 5. Method according to claim 1,wherein the time period is between 2 and 3 hours.
 6. Method according toclaim 1, wherein in step a) the temperature is 80° C., the concentrationof bisulfite is 5 M, the pH value of the solution is 5.5 and the timeperiod is between 2 and 3 hours.